The generation of biologically active lipid messengers by cellular biochemical processes is often under considerable biochemical regulation. However, biologically active lipids are also generated, in an unregulated mode, by the interaction of reactive oxygen species with lipid precursors present in the membranes of cells. The structural analysis and quantitation of these lipid mediators requires the development of sophisticated yet nonspecific analytical techniques that are capable of providing specific data for lipids while they are present in complex mixtures of closely related compounds. Mass spectrometry is such a tool. A major objective of this grant is to further develop sophisticated mass spectrometry including electrospray ionization and MALDI ionization techniques suitable for the qualitative and quantitative analysis of lipid mediators derived from cellular membranes following exposure to reactive oxygen species. The potential for mass spectrometry as a tool used for imaging lipids in tissues will also be investigated with the intent to apply mass spectrometric imaging to determine the precise localization of oxysterols in pulmonary airways following exposure of mice to environmentally relevant concentrations of ozone. One important specific goal is to understand the molecular basis underlying the toxicity of ozone in the lung. The oxidation of cholesterol by ozone and the formation of active oxysterols will be further examined. The reaction of ozone with plasmalogen glycerophospholipids will also be investigated in studies to test the hypothesis that vinyl ether phospholipids serve an important role in protecting cells from potentially toxic reactions of ozone following reactions with other endogenous lipids. The oxidation of phosphatidylserine and glycerophosphoethanolamine lipids during the process of cellular apoptosis will also be investigated. The specific goal will be to identify and structurally characterize oxidized phosphatidylserine and probe the mechanism of formation and potential biological functions of these oxidized lipids including the role they play in apoptosis. The studies in this portion of the program project grant complements the other projects in the broad sense of understanding mechanisms of lipid mediator production, regulation, and action.